WO2012172473A1 - Cysteine protease inhibitors - Google Patents

Abstract

Compounds of the formula (I) wherein One of A¹ and A² is N-CH₃ and the other is CH; R¹ is C₁-C₆alkyl, C₁-C₆haloalkyl, C₃-C₆cycloalkyl or oxetan-3-yl, wherein C₃-C₆cycloalkyl is optionally substituted with one, two or three fluoro or with CF₃; R^2a and R^2b are independently selected from H, halo, C₁-C₄alkyl, C₁-C₄haloalkyl and C₁- C₄alkoxy; R³ is CH₃ or F; n is 1, 2, 3 or 4; or a pharmaceutically acceptable salt, hydrate or N-oxide thereof for the use in the prophylaxis and/or treatment of a disorder characterised by inappropriate expression or activation of cathepsin S.

Description

Cysteine Protease Inhibitors

Technical Field

This invention relates to inhibitors of cathepsin S, and their use in the treatment of disorders involving cathepsin S such as autoimmune disorders, allergy and chronic pain conditions.

Background to the invention

The papain superfamily of cysteine proteases are widely distributed in diverse species including mammals, invertebrates, protozoa, plants and bacteria. A number of mammalian cathepsin enzymes, including cathepsins B, F, H, K, L, O, S, and W, have been ascribed to this

superfamily, and inappropriate regulation of their activity has been implicated in a number of metabolic disorders including arthritis, muscular dystrophy, inflammation, glomerulonephritis and tumour invasion. Pathogenic cathepsin like enzymes include the bacterial gingipains, the malarial falcipains I, II, III et seq and cysteine proteases from Pneumocystis carinii,

Trypanosoma cruzei and brucei, Crithidia fusiculata, Schistosoma spp.

In WO 97/40066, the use of inhibitors against Cathepsin S is described. The inhibition of this enzyme is suggested to prevent or treat disease caused by protease activity. Cathepsin S is a highly active cysteine protease belonging to the papain superfamily. Its primary structure is 57%, 41 % and 45% homologous with human cathepsin L and H and the plant cysteine protease papain respectively, although only 31 % homologous with cathepsin B. It is found mainly in B cells, dendritic cells and macrophages and this limited occurrence suggests the potential involvement of this enzyme in the pathogenesis of degenerative disease. Moreover, it has been found that destruction of li by proteolysis is required for MHC class II molecules to bind antigenic peptides, and for transport of the resulting complex to the cell surface. Furthermore, it has been found that Cathepsin S is essential in B cells for effective li proteolysis necessary to render class II molecules competent for binding peptides. Therefore, the inhibition of this enzyme may be useful in modulating class ll-restricted immune response (WO 97/40066).

Cathepsin S is also produced by inflammatory cells, such as dendritic cells, B cells and macrophages. It is involved in the pathology of several conditions including atherosclerosis and a condition known as abdominal aortic aneurysm (AAA).

The extracellular modelling of the cells is a continuously ongoing process to repair damaged arterial wall, wherein cathepsin S plays a role of degrading the extracellular matrix proteins such as elastin and collagen that make up the arterial wall. Occasionally the endothelial cells may malfunction, e.g. due to factors such as high levels of cholesterol, stress, overall health and genetics. This malfunction leads to the production and recruitment of inflammatory cells from the blood that penetrate the arterial wall to protect from damage and the inflammatory cells ultimately produce cathepsin S. The imbalance in the remodelling may lead to more proteolytic degradation which in turn may lead to instability of plaque formed within the arterial wall. Too much plaque instability could result in plaque rupture and potentially thrombotic-related events.

Further, the extracellular matrix of the abdominal aorta may be weekend due to excess degradation leading AAA. Thus, inhibition of cathepsin S provides a means for treating atherosclerosis and AAA.

Other disorders in which cathepsin S is implicated are asthma, chronic obstructive pulmonary disease, endometriosis and chronic pain.

Brief Description of the Invention

According to a first asp pound of the formula I:

(I) wherein

one of A¹ and A² is N-CH₃ and the other is CH;

R¹ is CrC₆alkyl, CrC₆haloalkyl, C₃-C₆cycloalkyl or oxetan-3-yl, wherein C₃-C₆cycloalkyl is optionally substituted with one, two or three fluoro or with CF₃;

R^2a and R^2b are independently selected from H, halo, CrC₄alkyl, Ci-C₄haloalkyl, Ci-C₄alkoxy; R³ is CH₃ or F;

n is 1 , 2, 3 or 4

or a pharmaceutically acceptable salt, hydrate or N-oxide thereof

Typical embodiments of the invention include compounds wherein n is 1 or 2, i.e. compounds having a 1 ,1 - cydopropylenyl or 1 ,1 -cyclobutylenyl ring, thus providing compounds according to formula la or lb respectively.

In some embodiments of the invention the 1 ,1 -cycloalkylenyl ring is unsubstituted, i.e. R^2a and R^2b are both H. Typically according to this embodiment, n is 1 or 2, thus providing compounds of the formulae la' and lb' respectively.

In some embodiments of the invention where n is 2, the 1 ,1 -cyclobutylenyl ring is substituted with one or two substituents, i.e. at least one of R^2a and R^2b is halo, CrC₄alkyl, d-C₄haloalkyl or CrC₄alkoxy. Typically according to these embodiments, the substituent(s) is/are located in the 2-position of the cyclobutylenyl ring, thus providing compounds according to formula Ic:

Typical embodiments where n is 2, include compounds wherein one of R and R is H, and the other is CI, F, CF₃ or MeO.

In a preferred embodiment where n is 2, one of R^2a and R^2b is H, and the other is F. Specially preferred according to this embodiment are compounds having the stereochemistry shown in formula Id:

In some embodiments where n is 2, R and R are both F, thus providing compounds according to formula le:

In other embodiments where n is 2, one of R and R is H, and the other is F, CF₃ or MeO. In some embodiments of the invention, R³ is CH₃. In other embodiments of the invention, R³ is F.

One embodiment of the invention include compounds wherein R¹ is CrC₆alkyl, such as ethyl, isopropyl or tert. butyl.

Another embodiment includes compounds wherein R¹ is CrC₆haloalkyl, such as chloroalkyi or fluoroalkyl. Typically according to this embodiment, R¹ is mono, di or tri fluoroCrC₆alkyl, such as monofluoropropyl, difluoropropyl, trifluoropropyl or trifluorobutyl.

In one embodiment of the invention compounds are included wherein R¹ is C₃-C₆cycloalkyl, which is optionally substituted with one two or three fluoro or with CF₃. Typically according to this embodiment, R¹ is cyclopropyl or cyclobutyl, or cyclopropyl or cyclobutyl substituted with one or two fluoro.

As stated above, one of A¹ and A² is N-CH₃ and the other is CH, i.e. the invention includes compounds of formulae If and Ig:

In typical embodiments of the invention, A¹ is N-CH₃, A² is CH and n is 1 or 2, thus providing compounds of formulae Ih and li respectively:

In further typical embodiments of the invention, A¹ is CH, A² is N-CH₃ and n is 1 or 2, thus providing compounds of formulae Ij and Ik respectively:

The compounds of formula I are characterised by various advantageous pharmaceutical properties and exhibit at least one improved property in view of the compounds of the prior art. In particular, the inhibitors of the present invention are superior in one or more of the following pharmacological related properties, i.e. potency, decreased cytotoxicity, improved

pharmacokinetics, acceptable dosage and pill burden.

Without in any way wishing to be bound by theory, or the ascription of tentative binding modes for specific variables, P1 , P2 and P3 as used herein are provided for convenience only and have their conventional meanings and denote those portions of the inhibitor believed to fill the S1 , S2 and S3 subsites respectively of the enzyme, where S1 is adjacent the cleavage site and S3 remote from the cleavage site.

A further aspect of the invention comprises a method employing the compounds of formula I or any subgroup of formula I for the prophylaxis or treatment of diseases caused by aberrant expression or activation of cathepsin, i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.

In a preferred aspect, the invention concerns a method employing the compounds of formula I or any subgroup thereof as specified herein, for the treatment of diseases caused by aberrant expression or activation of cathepsin, i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily. A further aspect of the invention provides the use of the compounds of formula I or any subgroup of formula I for the prophylaxis or treatment of diseases caused by aberrant expression or activation of cathepsin, i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.

In a preferred aspect, the invention concerns the use of compounds of formula I or any subgroup thereof as specified herein, for the treatment of diseases caused by aberrant expression or activation of cathepsin, i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.

A further aspect of the invention provides the use of the compounds of formula I or any subgroup of formula I for the manufacture of a medicament for the prophylaxis or treatment of diseases caused by aberrant expression or activation of cathepsin S, i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.

In a preferred aspect, the invention concerns the use of compounds of formula I or any subgroup of formula I as specified herein, for the manufacture of a medicament for the treatment of diseases caused by aberrant expression or activation of cathepsin S, i.e. diseases or conditions alleviated or modified by inhibition of cathepsin S, preferably without substantial concomitant inhibition of other members of the papain superfamily.

Examples of diseases or conditions for which the compounds of the invention may be useful include those enumerated in WO 97/40066, such as autoimmune diseases, allergies, such as asthma and hay fever, multiple sclerosis, rheumatoid arthritis and the like. A further example is endometriasis, and especially chronic pain, as disclosed in WO03/20287. Further examples are atherosclerosis, plaque instability and abdominal aortic aneurysm (AAA).

The invention further provides the use of the compounds of formula I or any subgroup of formula I in therapy and in the manufacture of a medicament for the treatment of diseases or conditions alleviated or moderated by inhibition of cathepsin S.

A further aspect of the present invention provides a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers, diluents or excipients, and optionally one or more other therapeutic agents.

In one series of embodiments, the methods are employed to treat mammals, particularly humans at risk of, or afflicted with, autoimmune disease. By autoimmunity is meant the phenomenon in which the host's immune response is turned against its own constituent parts, resulting in pathology. Many human autoimmune diseases are associated with certain class II MHC-complexes. This association occurs because the structures recognized by T cells, the cells that cause autoimmunity, are complexes comprised of class II MHC molecules and antigenic peptides. Autoimmune disease can result when T cells react with the host's class II MHC molecules when complexed with peptides derived from the host's own gene products. If these class II MHC/antigenic peptide complexes are inhibited from being formed, the autoimmune response is reduced or suppressed. Any autoimmune disease in which class II MHC/antigenic complexes play a role may be treated according to the methods of the present invention.

Such autoimmune diseases include, e.g., juvenile onset diabetes (insulin dependent), multiple sclerosis, pemphigus vulgaris, Graves' disease, myasthenia gravis, systemic lupus

erythematosus, rheumatoid arthritis and Hashimoto's thyroiditis.

In another series of embodiments, the methods are employed to treat mammals, particularly humans, at risk of, or afflicted with, allergic responses. By "allergic response" is meant the phenomenon in which the host's immune response to a particular antigen is unnecessary or disproportionate, resulting in pathology. Allergies are well known in the art, and the term

"allergic response" is used herein in accordance with standard usage in the medical field.

Examples of allergies include, but are not limited to, allergies to pollen, "ragweed," shellfish, domestic animals (e.g., cats and dogs), bee venom, house dust mite allergens and the like. Another particularly contemplated allergic response is that which causes asthma. Allergic responses may occur, in man, because T cells recognize particular class II MHC/antigenic peptide complexes. If these class II MHC/antigenic peptide complexes are inhibited from being formed, the allergic response is reduced or suppressed. Any allergic response in which class II MHC/antigenic peptide complexes play a role may be treated according to the methods of the present invention. Immunosuppression by the methods of the present invention will typically be a prophylactic or therapeutic treatment for severe or life-threatening allergic responses, as may arise during asthmatic attacks or anaphylactic shock. Preferably, the treatment is a therapeutic treatment. In another series of embodiments, the methods are employed to treat mammals, particularly humans, which have undergone, or are about to undergo, an organ transplant or tissue graft. In tissue transplantation (e.g., kidney, lung, liver, heart) or skin grafting, when there is a mismatch between the class II MHC genotypes (HLA types) of the donor and recipient, there may be a severe "allogeneic" immune response against the donor tissues which results from the presence of non-self or allogeneic class II MHC molecules presenting antigenic peptides on the surface of donor cells. To the extent that this response is dependent upon the formation of class II MHC/antigenic peptide complexes, inhibition of cathepsin S may suppress this response and mitigate the tissue rejection. An inhibitor of cathepsin S can be used alone or in conjunction with other therapeutic agents, e.g., as an adjunct to cyclosporin A and/or antilymphocyte gamma globulin, to achieve immunosuppression and promote graft survival. Preferably, administration is accomplished by systemic application to the host before and/or after surgery. Alternatively or in addition, perfusion of the donor organ or tissue, either prior or subsequent to transplantation or grafting, may be effective.

The above embodiments have been illustrated with an MHC class II mechanism but the invention is not limited to this mechanism of action. Suppression of cathepsin S as a treatment of COPD or chronic pain may not, for example, involve MHC class II at all.

A related aspect of the invention is directed to a method of treating a patient undergoing a therapy wherein the therapy causes an immune response, preferably a deleterious immune response, in the patient comprising administering to the patient a compound of Formula I or a pharmaceutically acceptable salt, n-oxide or hydrate thereof. Typically, the immune response is mediated by MHC class II molecules. The compound of this invention can be administered prior to, simultaneously, or after the therapy. Typically, the therapy involves treatment with a biologic, such as a protein, preferably an antibody, more preferably a monoclonal antibody. More preferrably, the biologic is Remicade®, Refacto®, ReferonA®, Factor VIII, Factor VII,

Betaseron®, Epogen®, Enbrel®, Interferon beta, Botox®, Fabrazyme®, Elspar®, Cerezyme®, Myobloc®, Aldurazyrne®, Verluma®, Interferon alpha, Humira®, Aranesp®, Zevalin® or OKT3. Alternatively the treatment involves use of heparin, low molecular weight heparin, procainamide or hydralazine.

Assays for the assessment of cathepsin S inhibitors in the treatment of chronic pain, including neuropathic or inflammatory pain are as described in WO03/20287.

Currently preferred indications treatable in accordance with the present invention include: Psoriasis;

Pruritus;

Autoimmune indications, including idiopathic thrombocytopenic purpura (ITP), rheumatoid arthritis (RA), multiple schlerosis (MS), myasthenia gravis (MG), Sjogrens syndrome, Grave's disease and systemic lupus erythematosis (SLE);

Non-automimmune indications include allergic rhinitis, asthma, artherosclerosis, chronic obstructive pulmonary disease (COPD) and chronic pain.

The compounds of the invention can form salts which form an additional aspect of the invention. Appropriate pharmaceutically acceptable salts of the compounds of the invention include salts of organic acids, especially carboxylic acids, including but not limited to acetate, trifluoroacetate, lactate, gluconate, citrate, tartrate, maleate, malate, pantothenate, isethionate, adipate, alginate, aspartate, benzoate, butyrate, digluconate, cyclopentanate, glucoheptanate, glycerophosphate, oxalate, heptanoate, hexanoate, fumarate, nicotinate, palmoate, pectinate, 3-phenylpropionate, picrate, pivalate, propionate, tartrate, lactobionate, pivolate, camphorate, undecanoate and succinate, organic sulphonic acids such as methanesulphonate, ethanesulphonate,

2-hydroxyethane sulphonate, camphorsulphonate, 2-naphthalenesulphonate,

benzenesulphonate, p-chlorobenzenesulphonate and p-toluenesulphonate; and inorganic acids such as hydrochloride, hydrobromide, hydroiodide, sulphate, bisulphate, hemisulphate, thiocyanate, persulphate, phosphoric and sulphonic acids.

The compounds of the invention may in some cases be isolated as the hydrate. Hydrates are typically prepared by recrystallisation from an aqueous/organic solvent mixture using organic solvents such as dioxin, tetrahydrofuran or methanol. Hydrates can also be generated in situ by administration of the corresponding keton to a patient.

The N-oxides of compounds of the invention can be prepared by methods known to those of ordinary skill in the art. For example, N-oxides can be prepared by treating an unoxidized form of the compound of the invention with an oxidizing agent (e.g., trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta-chloroperoxybenzoic acid, or the like) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at

approximately 0 °C. Alternatively, the N-oxides of the compounds of the invention can be prepared from the N-oxide of an appropriate starting material.

Compounds of the invention in unoxidized form can be prepared from N-oxides of the corresponding compounds of the invention by treating with a reducing agent (e.g., sulphur, sulphur dioxide, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus dichloride, tribromide, or the like) in an suitable inert organic solvent (e.g., acetonitrile, ethanol, aqueous dioxane, or the like) at 0 to 80 °C.

The present invention also includes isotope-labelled compounds of formula I or any subgroup of formula I, wherein one or more of the atoms is replaced by an isotope of that atom, i.e. an atom having the same atomic number as, but an atomic mass different from, the one(s) typically found in nature. Examples of isotopes that may be incorporated into the compounds of formula I or any subgroup of formula I, include but are not limited to isotopes of hydrogen, such as ²H and ³H (also denoted D for deuterium and T for tritium respectively), carbon, such as ¹¹C, ¹³C and ¹⁴C, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵0, ¹⁷0 and ¹⁸0, phosphorus, such as ³¹P and ³²P, sulphur, such as ³⁵S, fluorine, such as ¹⁸F, chlorine, such as ³⁶CI, bromine such as ⁷⁵Br, ⁷⁶Br, ⁷⁷Br and ⁸²Br, and iodine, such as ¹²³l, ¹²⁴l, ¹²⁵l and ¹³¹l.

The choice of isotope included in an isotope-labelled compound will depend on the specific application of that compound. For example, for drug or substrate tissue distribution assays, compounds wherein a radioactive isotope such as ³H or ¹⁴C is incorporated will generally be most useful. For radio-imaging applications, for example positron emission tomography (PET) a positron emitting isotope such as ¹¹C, ¹⁸F, ¹³N or ¹⁵0 will be useful. The incorporation of a heavier isotope, such as deuterium, i.e. ²H, may provide greater metabolic stability to a compound of formula I or any subgroup of formula I, which may result in, for example, an increased in vivo half life of the compound or reduced dosage requirements. For example, ²H isotope(s) are typically incorporated at position(s) disposed to metabolic liability. In the compounds of the present invention, suitable positions for incorporation of ²H isotopes are e.g. as substituents to the 1 ,1 -cycloalkylene group, i.e. one or both of R^2a and R^2b is ²H.

Isotopically labelled compounds of formula I or any subgroup of formula I can be prepared by processes analogous to those described in the Schemes and/or Examples herein below by using the appropriate isotopically labelled reagent or starting material instead of the

corresponding non-isotopically labelled reagent or starting material, or by conventional techniques known to those skilled in the art.

It should be noted that the radical positions on any molecular moiety used in the definitions may be anywhere on such moiety as long as it is chemically stable.

As used herein, the following terms have the meanings as defined below:

"C_m-C_nalkyl" used herein represents a straight or branched alkyl radical having the number of carbon atoms designated, e.g. CrC₄alkyl means an alkyl radical having from 1 to 4 carbon atoms. Preferred alkyl radicals for use in the present invention are CrC₄alkyl and includes methyl, ethyl, n-propyl, isopropyl, t-butyl, n-butyl and isobutyl. Methyl and t-butyl are typically preferred. CrC₆alkyl has a corresponding meaning, including also all straight and branched chain isomers of pentyl and hexyl. The expression "C_m-C_nalkyl" may be used on its own or in composite expressions such as C_m-C_nhaloalkyl, C_m-C_nalkylcarbonyl, C_m-C_nalkylamine etc.

The term "Me" means methyl, "MeO" means methoxy, "Et" means ethyl and "Ac" means acetyl.

"Ci-C₄haloalkyl" as used herein represents CrC₄alkyl, wherein at least one C atom is substituted with a halogen, preferably chloro or fluoro. Trifluoromethyl is typically preferred

"C_m-C_nalkoxy" as used herein represents a radical C_m-C_nalkyl-0 wherein C_m-C_nalkyl is as defined above. Typical C_m-C_nalkoxy includes Ci-C₄alkoxy such as methoxy, ethoxy, n-propoxy, isopropoxy, t-butoxy, n-butoxy and isobutoxy. Methoxy and isopropoxy are typically preferred. CrC₆alkoxy has a corresponding meaning, expanded to include all straight and branched chain isomers of pentoxy and hexoxy.

"C_m-C_nhaloalkoxy" as used herein represents C_m-C_nalkoxy wherein at least one C-atom is substituted with one or more halogen atom(s), typically chloro or fluoro. In many cases trifluoromethyl is preferred. Among the C_m-C_nhaloalkoxy Ci-C₄haloalkoxy are preferred.

"C₃-C_nCycloalkyl" as used herein represents to a cyclic monovalent alkyl radikal having the number of carbon atoms indicated, e.g. C₃-C₆cycloalkyl means a cyclic monovalent alkyl radical having from 3 to 6 carbon atoms. Preferred cycloalkyl radicals for use in the present invention are C₃-C₄alkyl i.e. cyclopropyl and cyclobutyl.

"C₃-C_nCycloalkylenyl" as used herein represents to a divalent cycloalkyl radikal having the number of carbon atoms indicated. Preferred cycloalkylenyl radicals for use in the present invention are C₃-C₄alkylenyl i.e. cyclopropylenyl and cyclobutylenyl.

"C_m-C_nalkoxycarbonyl" means a radical C_m-C_nalkyl-0-C(=0).

The compounds of the invention include a number of handles such as OH, NH or COOH groups to which conventional prodrug moieties can be applied. Prodrugs are typically hydrolysed in vivo to release the parent compound in the plasma, liver or intestinal wall. Favoured prodrugs are esters of hydroxyl groups such as a phenolic hydroxyl group at R⁴, or amine functions such as a sulphonamide amine function. Preferred pharmaceutically acceptable esters include those derived from Ci-C₆ carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubstituted or substituted with substituents broadly as described for R^1a, typically 1 -3 halo (e.g. F), C C₄alkyl (e.g. Me), C C₄haloalkyl (e.g. CF₃) or C C₄alkyloxy (e.g. MeO) groups. Favoured sulphonamide prodrugs include aminoacyls derived from Ci-C₆ carboxylic acids such as acetyl or pivaloyl or optionally substituted benzoic acid esters, preferably unsubstituted or substituted with substituents broadly as described for variable R^1a, typically 1 -3 halo (e.g. F), Ci-C₄alkyl (e.g. Me), Ci-C₄haloalkyl (e.g. CF₃) or Ci-C₄alkyloxy (e.g. MeO) groups.

Unless otherwise mentioned or indicated, the chemical designation of a compound

encompasses the mixture of all possible stereochemically isomeric forms, which said compound may possess. Said mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of said compound. All stereochemically isomeric forms of the compounds of the present invention both in pure form or mixed with each other are intended to be embraced within the scope of the present invention.

Pure stereoisomeric forms of the compounds and intermediates as mentioned herein are defined as isomers substantially free of other enantiomeric or diastereomeric forms of the same basic molecular structure of said compounds or intermediates. In particular, the term

"stereoisomerically pure" concerns compounds or intermediates having a stereoisomeric excess of at least 80% (i.e. minimum 90% of one isomer and maximum 10% of the other possible isomers) up to a stereoisomeric excess of 100% (i.e. 100% of one isomer and none of the other), more in particular, compounds or intermediates having a stereoisomeric excess of 90% up to 100%, even more in particular having a stereoisomeric excess of 94% up to 100% and most in particular having a stereoisomeric excess of 97% up to 100%. The terms "enantiomerically pure" and "diastereomerically pure" should be understood in a similar way, but then having regard to the enantiomeric excess, and the diastereomeric excess, respectively, of the mixture in question.

Compounds of the invention can be prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers and recovering the optically pure enantiomer. While resolution of enantiomers can be carried out using covalent diasteromeric derivatives of compounds of Formula I, dissociable complexes are preferred (e.g., crystalline; diastereoisomeric salts). Diastereomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by taking advantage of these dissimilarities. The diastereomers can be separated by chromatography, for example HPLC or, preferably, by separation/resolution techniques based upon differences in solubility. The optically pure enantiomer is then recovered, along with the resolving agent, by any practical means that would not result in racemization. A more detailed description of the techniques applicable to the resolution of stereoisomers of compounds from their racemic mixture can be found in Jean Jacques Andre Collet, Samuel H. Wilen, Enantiomers, Racemates and

Resolutions, John Wiley & Sons, Inc. (1981 ).

While it is possible for the active agent to be administered alone, it is preferable to present it as part of a pharmaceutical formulation. Such a formulation will comprise the above defined active agent together with one or more acceptable carriers/excipients and optionally other therapeutic ingredients. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

The formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, but preferably the formulation is an orally administered formulation. The formulations may conveniently be presented in unit dosage form, e.g. tablets and sustained release capsules, and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the above defined active agent with the carrier. In general, the formulations are prepared by uniformly and intimately bringing into association the active agent with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The invention extends to methods for preparing a

pharmaceutical composition comprising bringing a compound of Formula I or its

pharmaceutically acceptable salt in conjunction or association with a pharmaceutically acceptable carrier or vehicle. If the manufacture of pharmaceutical formulations involves intimate mixing of pharmaceutical excipients and the active ingredient in salt form, then it is often preferred to use excipients which are non-basic in nature, i.e. either acidic or neutral.

Formulations for oral administration in the present invention may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active agent; as a powder or granules; as a solution or a suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water in oil liquid emulsion and as a bolus etc.

With regard to compositions for oral administration (e.g. tablets and capsules), the term suitable carrier includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of wintergreen, cherry flavouring or the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active agent.

Other formulations suitable for oral administration include lozenges comprising the active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active agent in an inert base such as gelatine and glycerine, or sucrose and acacia; and mouthwashes comprising the active agent in a suitable liquid carrier.

As with all pharmaceuticals, the appropriate dosage for the compounds or formulations of the invention will depend upon the indication, the severity of the disease, the size and metabolic vigour and the patient, the mode of administration and is readily determined by conventional animal trials. Dosages providing intracellular (for inhibition of physiological proteases of the papain superfamily) concentrations of the order 0.01 -100 μΜ, more preferably 0.01 -10 μΜ, such as 0.1 -5 μΜ are typically desirable and achievable.

Compounds of the invention are prepared by a variety of solution and solid phase chemistries. A typical first step is the preparation of a P1 building block of the formula II

where R and R are as defined above and PG is a conventional N protecting group such as Boc, CBz or Fmoc. These building blocks are novel and constitute a further aspect of the invention. B ilding blocks of formula II are typically prepared as described in scheme 1 below.

1d 1e

Scheme 1

A suitable starting material is an N-protected cycloalkyl amino acid, of which several are available commercially or can be prepared as shown in the following Examples or as described by Allan et al. in J. Med. Chem., 1990 33(10) 2905-2915.

The carboxylic acid (1 a) is transformed via a Weinreb synthesis to a Ν,Ο-dimethylhydroxamic acid (1 b) which provides the corresponding aldehyde (1 c). The aldehyde may also be accessed by reduction of the carboxylic function of a cycloalkyl amino acid followed by oxidation under Dess Martin conditions. The aldehyde (1 c) can be subsequently reacted with t. butyl isocyanide or equivalent in a Passerini reaction to afford the ohydroxy amide (1 d). Subsequent hydrolysis of the amide then provides the required ohydroxycarboxylic acid P1 building block (1 e).

Generally the strongly acidic conditions required to hydrolyse the amide also lead to loss of the NBoc protection, if used. Hence, the amine can be used directly to couple to a P2 building block or else if it needs to be stored, the amine can be reprotected.

The P1 building block thus afforded is then modified/extended at the C and N termini to provide compounds of formula I, as illustrated in Scheme 2 below.

Scheme 2

Typically the C terminus of the building block of formula II is extended at the C-termini by reaction with with N-methyl pyrazole or imidazole amine using any suitable conventional peptide coupling stategy. The thus prepared P1-prime side unit (2a) is thereafter deprotected at the N terminus and elongated with the P2 and subsequently P3 building blocks using conventional peptide chemistries. For example a P2 residue can be introduced via BocP2-OH using standard coupling conditions such as HATU, DIPEA in DMF. The terminal Boc protection is again removed with acetyl chloride in methanol or equivalent and the P3 residue introduced via P3-OH using standard coupling conditions such as HATU, DIPEA in DMF. Alternatively, A P3- P2-building block may be prepared separately and added directly to the P1 -prime side-building block (2a) in one step, thus affording the dipeptide derivative (2c). Oxidation of the ohydroxy amide to the corresponding a-keto amide (2d) is then effected using any convenient oxidation method known in the art, such as Dess Martin oxidation or Moffat oxidation or the like.

An extensive range of appropriately protected L-amino acids suitable for P2 building blocks and carboxylic acids, carboxylic acid halides and carbamoyl halides suitable for P3 building blocks are commercially available or accessed by simple chemistries or as shown in WO06/064286. The P3 and P2 building blocks may alternatively be coupled first and then reacted with the P1 - prime side unit.

Elongation is typically carried out in the presence of a suitable coupling agent e.g.,

benzotriazole-1 -yloxytrispyrrolidinophosphonium hexafluorophosphate (PyBOP), O- benzotriazol-l-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU), 0-(7- azabenzotriazol-1 -yl)-1 ,1 ,3,3-tetramethyl-uronium hexafluorophosphate (HATU), 1 -(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDO), or 1 ,3-dicyclohexyl

carbodiimide (DCC), optionally in the presence of l-hydroxybenzotriazole (HOBT), and a base such as Ν,Ν-diisopropylethylamine, triethylamine, N-methylmorpholine, and the like. The reaction is typically carried out at 20 to 30 °C, preferably at about 25 °C, and requires 2 to 24 h to complete. Suitable reaction solvents are inert organic solvents such as halogenated organic solvents (e.g., methylene chloride, chloroform, and the like), acetonitrile, N,N- dimethylformamide, ethereal solvents such as tetrahydrofuran, dioxane, and the like.

Alternatively, the above elongation coupling step can be carried out by first converting the P3/P2 building block into an active acid derivative such as succinimide ester and then reacting it with the P1 amine. The reaction typically requires 2 to 3 h to complete. The conditions utilized in this reaction depend on the nature of the active acid derivative. For example, if it is an acid chloride derivative, the reaction is carried out in the presence of a suitable base (e.g.

triethylamine, diisopropylethylamine, pyridine, and the like). Suitable reaction solvents are polar organic solvents such as acetonitrile, Ν,Ν-dimethylformamide, dichloromethane, or any suitable mixtures thereof.

The term "N-protecting group" or "N-protected" as used herein refers to those groups intended to protect the N-terminus of an amino acid or peptide or to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis" (John Wiley & Sons, New York, 1981 ), which is hereby incorporated by reference. N-protecting groups include acyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoracetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, a-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4- bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonyl groups such as benzenesulfonyl, p- toluenesulfonyl, and the like, carbamate forming groups such as benzyloxycarbonyl, p- chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,

2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,

3,4,5-trimethoxybenzyloxycarbonyl, 1 -(p-biphenylyl)-1 -methylethoxycarbonyl, a,a-dimethyl-3,5- dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl, t-butoxycarbonyl,

diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl,

cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and silyl groups such as trimethylsilyl and the like. Favoured N-protecting groups include formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl (bz), t-butoxycarbonyl (BOC) and benzyloxycarbonyl (Cbz). Hydroxy and/or carboxy protecting groups are also extensively reviewed in Greene ibid and include ethers such as methyl, substituted methyl ethers such as methoxymethyl,

methylthiomethyl, benzyloxymethyl, t-butoxymethyl, 2-methoxyethoxymethyl and the like, silyl ethers such as trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS) tribenzylsilyl, triphenylsilyl, t- butyldiphenylsilyl triisopropyl silyl and the like, substituted ethyl ethers such as 1 -ethoxymethyl, 1 -methyl-1 -methoxyethyl, t-butyl, allyl, benzyl, p-methoxybenzyl, dipehenylmethyl,

triphenylmethyl and the like, aralkyi groups such as trityl, and pixyl (9-hydroxy-9-phenylxanthene derivatives, especially the chloride). Ester hydroxy protecting groups include esters such as formate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate, pivaloate,

adamantoate, mesitoate, benzoate and the like. Carbonate hydroxy protecting groups include methyl vinyl, allyl, cinnamyl, benzyl and the like.

Detailed Description of the Embodiments

Various embodiments of the invention will now be described by way of illustration only with reference to the following Examples.

In the examples below, the following systems are typically employed:

Nuclear Magnetic Resonance (NMR) spectra were recorded on a Varian Gemini 7 Tesla 300 MHz instrument, or a Bruker Avance 400 MHz instrument in the solvent indicated. Chemical shifts are given in ppm down- and upfield from tetramethylsilane (TMS). Resonance

multiplicities are denoted s, d, t, m, br and app for singlet, doublet, triplet, multiplet, broad and apparent, respectively. The Mass Spectrometry (MS) spectra were recorded on a Finnigan SSQ7000 TSP or a Finnigan SSQ710 DI/EI instrument. LC-MS was obtained with a Waters 2790 LC-system equipped with a Waters Xterra™ MS C₈ 2.5 μηη 2.1 x 30 mm column, a Waters 996 Photodiode Array Detector and a Micromass ZMD. High pressure liquid chromatography (HPLC) assays were performed using a Hewlett Packard 1 100 Series HPLC system equipped with a Zorbax column SB-C₈ 4.6 mmx15 cm. Column chromatography was performed using silica gel 60 (230-400 mesh ASTM, Merck) and thin layer chromatography (TLC) was performed on TLC precoated plates, silica gel 60 F₂₅4 (Merck). Preparation of Building Block 1 (BB1 ), a P1 building block

BB1 -C BB1

Step a) 2-(1 -((tert-butoxycarbonyl)amino)cvclobutyl)-2-hvdroxyacetic acid (BB1 -a)

To a solution of 1 -ie f-butoxycarbonylamino-cyclobutanecarboxylic acid (3 g, 13.94 mmol) in dry DMF (50 mL) was added Ν,Ο-dimethylhydroxylamine x HCI (1.36 g, 13.94 mmol) and DIEA (9.21 mL, 55.75 mmol). The reaction flask was cooled to 0 °C and after 10 minutes HATU (5.30 g, 13.94 mmol) was added to the solution (which turned yellow on addition). After 2 hrs the DMF was removed by rotary evaporation at reduced pressure. The residue was dissolved in EtOAc (100 mL) and washed twice with 10 % citric acid (aq) and saturated NaHC0₃(aq) solution. The organic phase was dried with Na₂S0₄, filtered and evaporated on silica. The product was purified by flash chromatography (heptane: ethyl acetate (1 :1 ) to give the product as a colourless oil that slowly crystallizes (3.13 g) in 87 % yield.

Step b) (l -Formyl-cvclobutvD-carbamic acid te f-butyl ester (BB1 -b)

LiAIH₄ (202 mg, 5.33 mmol) was added to a solution of the Weinreb amide BB1 -a (1 .10 g, 4.27 mmol) dissolved in dry diethyl ether (35 mL) at 0 °C. The solution was stirred at 15 minutes before the reaction was quenched with slow addition of potassium hydrogen tartaric acid (sat, aq) and stirred for 10 minutes. The solution was poured into a separatory funnel and the water phase was extracted with ethyl acetate twice. The combined organic phases were washed with 0.5 M HCI (3 times), NaHC0₃(aq) (2 times) and brine (1 time). The organic phase was dried with Na₂S0₄, filtered and evaporated on silica. The product was purified by flash chromatography (heptane: ethyl acetate (4:1→ 3:1 ) to give the product as white crystals (0.647 g) in 76 % yield.

Step c) [1 -(tert-Butylcarbamoyl-hvdroxy-methyl)-cvclobutyll-carbamic acid tert-butyl ester (BB1 - c)

BB1 -b, (1.75 g, 8.78 mmol) was dissolved in CH₂CI₂ (18 mL) and cooled in an ice bath, under inert gas. Pyridine (2.85 mL) was added, followed by t-butyl isocyanide (1 .50 mL, 13.3 mmol). Trifluoroacetic acid (1 .35 mL, 17.5 mmol) was then added dropwise over 30 min. The yellow solution was stirred at RT overnight. The mixture was concentrated, diluted with EtOAc (100 mL) and washed successively with 1 N HCI (50 mL), saturated NaHC0₃ (50 mL) and saturated NaCI (2 x 50 mL). Drying (Na₂S0₄) and concentration under vacuum. The afforded crude product was treated with THF (2.5 mL) and 1 M LiOH in 3/1 MeOH-water (2.5 mL) at RT. TLC (3/1 petroleum ether - EtOAc) showed complete ester hydrolysis after 15 min. After 45 min reaction time, 1 N HCI (2.5 mL), water (10 mL) and EtOAc (20 mL) were added, and the layers were separated. The organic phase was washed with saturated NaHC0₃ (20 mL) and then saturated NaCI (2 x 20 mL), dried (Na₂S0₄) and concentrated. Flash chromatography (75 g silica, 5/1 to 1/1 petroleum ether - EtOAc) gave a white solid ( 2.36 g, 89 %).

Step d (l -tert-Butoxycarbonylamino-cvclobutyD-hydroxy-acetic acid (BB1 )

BB1 -c (1 .30 g, 4.33 mmol) was refluxed with 6N HCI (40 mL) until amide hydrolysis was complete as monitored by LCMS. The mixture was evaporated, co-evaporating several times with water. 1 M NaOH (15 mL) was added to the residue and the basic solution was stirred under vacuum for 15 min. Boc₂0 (1 .92 g, 8.80 mmol) in dioxane (10 mL) was added, keeping pH at 10 - 1 1 , and the mixture was stirred at RT overnight. The mixture was diluted with water (50 mL), acidified with 1 N HCI to pH 3, in an ice bath, and then extracted with EtOAc (2 x 50 mL, then 30 mL). The organic phase was washed with saturated NaCI (50 mL), dried (Na₂S0₄) and evaporated to give crude P1 building block BB1 (0.649 g).

^1HNMR (400 MHz, d₆-DMSO) δ 6.88 (br s, 1 H), 4.15 (s, 1 H), 2.40 (br m, 2H), 1 .98 (br m, 2H), 1.80 (br m, 2H), 1 .35 (s, 9H); ms ES⁺ m/z 146 (100 %), 190 (50 %).

Preparation of Building Block 2 (BB2). a P1 building block

Step a) ((1 -Bromo-3-chloropropan-2-yloxy)methyl)benzene (BB2-a)

Epichlorohydrin (100 g, 1.08 mol) was added to a stirred mixture of benzyl bromide (185 g, 1.08 mol) and (1 .5 g) of mercurous chloride. The reaction mixture was heated for 12 h at 100 °C until TLC indicated formation of product. The product was isolated by column chromatography using petroleum ether as eluent. Yield; 148 g, 51 %.

Step b) 3-Benzyloxy-cvclobutane-1 ,1 -dicarboxylic acid diethyl ester (BB2-b)

To a stirred suspension of sodium hydride (22.5 g, 0.562 mol) in 800 mL of dry dioxane, was added diethyl malonate (90 g, 0.562 mol) drop-wise over 20 min. After this addition was complete, BB2-a (148 g, 0.56 mol) was added drop-wise over 20 min. The mixture was then heated at reflux for 24 hr. After cooling to room temperature, sodium hydride (22.5 g, 0.562 mol) in a little dioxane (~ 20 mL) was added to the mixture and heating at reflux was continued for an additional 48 hr. The solvent was partially removed under reduced pressure and water (800 mL) was added. The mixture was then extracted with EtOAc (500 mL x 3), the organic extracts were dried (Na₂S0₄) and concentrated in vacuo and the residue was purified by column

chromatography using petroleum ether: EtOAc (10 %) which gave the title compound (100 g, 58 %).

Step c) Diethyl 3-hydroxycvclobutane-1 ,1-dicarboxylate (BB2-c)

To a solution of compound BB2-b (40 g) in EtOH (500 mL) was added 10 % palladium on charcoal (4 g) and the mixture was hydrogenated for 3.5 hours at 50 psi at room temperature. The catalyst was removed by filtration and the filter washed with ethyl acetate and EtOH. The solvents were removed under reduced pressure and theafforded residue was purified by silica gel chromatography eluted with hexane/ EtOAc, which gave the title compound (18 g, 64 %).

Step d) Diethyl 3-oxocvclobutane-1 ,1 -dicarboxylate (BB2-d)

To a solution of compound BB2-C (18 g, 0.0833 mol) in DCM (200 mL) was added PCC

(37 g, 0.176 mol) and the mixture was stirred for four hours at room temperature. The solution was filtered through a silica gel column and the residue was washed with DCM/MeOH 98/2 and then filtered through a similar column. The combined fractions were evaporated under reduced pressure which gave the title compound (1 1 g, 62 %).

Step e) Diethyl 3,3-difluorocyclobutane-1 ,1 -dicarboxylate (BB2-e)

To a cooled solution of compound BB2-d (1 1 g, 0.0513 mol) in dry DCM (150 mL) was added drop-wise a solution of DAST (18.72 g, 0.1 16 mol) and the mixture was stirred at room temperature overnight. The mixture was added to ice water and was extracted three times with DCM. The solution was dried with sodium sulphate and evaporated under reduced pressure. The residue was purified by silica gel chromatography eluted with hexane/ EtOAc which gave the title compound (7.7 g, 64 %).

Step f) 1 -(Ethoxycarbonyl)-3,3-difluorocvclobutanecarboxylic acid (BB2-f)

Compound BB2-e (7.7 g, 0.0325 mol) was dissolved in ice cooled 0.5 M ethanolic potassium hydroxide solution (30 mL) and water (6 mL). The mixture was stirred at room temperature overnight. Water was added and most of the ethanol was removed under reduced pressure. The mixture was acidified with 2M HCI and extracted three times with EtOAc. The organic phase was dried with sodium sulphate and evaporated under reduced pressure which gave the title compound (5.8 g, 86 %).

Step g) Ethyl 1 -(tert-butoxycarbonylamino)-3,3-difluorocvclobutanecarboxylate (BB2-g)

To a solution of compound BB2-f (5.8 g, 0.0273 mol) in dry dioxane (100 mL) was added tert- butanol (24.4 mL), DPPA (7.87 g, 0.027 mol) and TEA (2.87 g, 0.0284 mol) and the mixture was refluxed for five hours. Ethyl acetate (about 200 mL) was added and the organic phase was washed twice with 5 % citric acid and saturated sodium hydrogen carbonate. The solution was dried and evaporated under reduced pressure. The afforded residue was purified by silica gel chromatography eluted with hexane/ EtOAc, which gave the title compound (4 g, 51 .4 %).

Step h) tert-Butyl 3,3-difluoro-1 -(hvdroxymethyl)cvclobutylcarbamate (BB2-h)

To a ice cooled solution of compound BB2-g (4 g , 14.3 mmol) in dry THF (100 mL) was slowly added a solution of 2M lithium borohydride (30 mL) and the mixture was allowed to warm up to room temperature. The mixture was stirred for three hours at room temperature. Ice water and 5 % citric acid were added and the mixture was extracted three times with DCM. The organic phase was dried (Na₂S0₄), filtered and evaporated under reduced pressure which gave the title compound (3.1 g, 91 %).

Step i) tert-Butyl 3,3-difluoro-1 -formylcvclobutylcarbamate (BB2-Q

To a solution of compound BB2-h (3.1 g, 0.0130 mol) in dry DCM (100 mL) was added Dess Martin Period inane (19.9 g, 0.0470 mol) and the mixture was stirred for three hours at room temperature. Ethyl acetate (200 mL) was added and the organic phase was washed twice with 10 % sodium thiosulphate solution, twice with 0.5 M NaOH and with brine. The organic phase was dried and evaporated under reduced pressure. The residue was purified by silica gel chromatography with hexane/ethyl acetate as eluent which gave the title compound (2.7 g, 87 %). Step i) tert-Butyl 1 -(2-(tert-butylamino)-1-hvdroxy-2-oxoethyl)-3,3-difluorocvclobutylcarbamate (BB2-I)

To a ice cooled solution of compound BB2-i (1.5 g, 0.0064 mol) in dry DCM (100 mL) was added tert-butylisocyanate (0.81 g, 0.009 mol) and pyridine (2.04 g, 0.027 mol). Trifluoroacetic acid (1.58 g, 0.015 mol) was added over a ten minutes period. The mixture was stirred for five hours at room temperature. Ethyl acetate was added and the organic phase was washed twice with 5 % citric acid and brine. The organic phase was evaporated and dissolved in dioxane (50 mL). 1 M LiOH solution (100 mL) was added and the mixture was stirred overnight at room temperature. 5 % Citric acid was added and the mixture was extracted three times with ethyl acetate. The organic phase was washed with brine, dried (Na₂S0₄), filtered and evaporated under reduced pressure. The afforded residue was purified by silica gel chromatography elutd with hexane/ EtOAc, which gave the title compound (1 .0 g, 46 %).

Step k) 2-(1 -(tert-Butoxycarbonylamino)-3,3-difluorocvclobutyl)-2-hvdroxyacetic acid (BB2) Compound BB2-j (1 g) was dissolved in 6N HCI (40 mL), and heated to reflux for 24 h after which TLC showed that the reaction had reached completion. The reaction mixture was concentrated in vacuo and residue was dissolved in THF; H₂0 (7; 3, 50 mL), and TEA (1 .8 mL, 0.012 mol) and Boc anhydride (2.6 g, 0.012 mol) were both added. The mixture was stirred at RT for 8 h when TLC confirmed the reaction had reached completion. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography using 5% methanol in chloroform which gave the title compound (0.6 g, 72 %).

^1H NMR (400 MHz, d₆-DMSO) δ 7.30 (br s, 1 H), 4.1 1 (s, 1 H), 2.90 (br m, 2H), 2.61 (br m, 2H), 1.35 (s, 9H); ms ES⁺ m/z 281 (100 %).

Preparation of building block 3, a P1 building block

BB3-g BB3-h ΒΒ3-Ϊ BB3 Step a) ((1 -Bromo-3-chloropropan-2-yloxy)methyl)benzene (BB3-a)

A mixture of benzyl bromide (46.0 g, 0.269 mol) and epichlorohydrin (24.9 g, 0.269 mol) and mercurous chloride (0.04 g, 0.085 mmol) was heated for 12 h at 150 °C. The crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 1 % EtOAc in pet ether) which afforded the title compound as a viscous liquid (50 g, 70 %).

Step b) Diethyl 3-(benzyloxy)cvclobutane-1 ,1 -dicarboxylate (BB3-b)

In a three-neck flask equipped with stirrer, additional funnel and reflux condenser was place NaH (4.6 g, 0.192 mol) in dry dioxane (150 mL). To this stirred reaction mixture, diethyl malonate (30.75 g, 0.192 mol) was added drop-wise over 30 min. After the addition was complete, compound BB3-a (50 g, 0.19 mol) was added drop-wise over a period of 30 min. The reaction mixture was refluxed for 24 h. After cooling to room temperature, NaH (4.6 g, 0.192 mol) and dry dioxane (40 mL) was added to the reaction mixture and further heated to reflux for another 48 h. The solvent was partially removed under reduced pressure and the mixture was treated with water (150 mL). The product was extracted with diethyl ether (3 x 100 mL), the organic layer was washed with brine and dried over anhydrous Na₂S0₄. Solvent was concentrated in vacuum and the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 2 % EtOAc in p. ether) which afforded the title compound as a viscous liquid (33 g, 57 %).

Step c) Diethyl 3-hydroxycvclobutane-1 ,1-dicarboxylate (BB3-c)

To a solution of compound BB3-b (33 g, 0.108 mol) in EtOH (300 mL) was added 10 % palladium on charcoal (10 g) and the mixture was hydrogenated for 48 h with 50 psi pressure at room temperature. The catalyst was removed by filtration through a Celite bed and washed thoroughly with EtOAc. The solvent was removed under reduced pressure. The product was purified by silica gel chromatography (silica gel 60-120 mesh, eluent 20 % EtOAc in pet ether) which afforded the title compound as a viscous liquid (12 g, yield 51 %).

Step d) Diethyl 3-fluorocvclobutane-1 ,1 -dicarboxylate (BB3-d)

Compound BB3-C (0.8 g, 0.0037 mol) was dissolved in dry DCM (16 mL) and cooled to 0 °C. DAST (1 .8 g, 1 1 mmol) was added drop-wise to the cold solution. The reaction mixture was warmed to room temperature stirred for 12 h. The reaction mixture was quenched with cold saturated NaHC0₃ solution. The crude product was extracted with DCM (100 mL). The organic layer was washed with 10 % NaHC0₃ solution, water followed by brine and dried over anhydrous Na₂S0₄. Solvent was concentrated in vacuum and the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 1 -2 % EtOAc in pet ether) which afforded the title compound as a pale yellow liquid (460 mg, yield 57 %). Step e) 1 -(Ethoxycarbonyl)-3-fluorocvclobutanecarboxylic acid (BB3-e)

Compound BB3-d (0.46 g, 0.0021 mol) was dissolved in ice cooled 0.5M potassium hydroxide solution in EtOH (4.2 mL) and water (1 .4 mL). The mixture was stirred at room temperature overnight. Water was added and most of the ethanol was removed under reduced pressure. The mixture was acidified with 2N HCI and extracted with EtOAc (3 x 50 mL). The organic phase was dried over anhydrous Na₂S0₄. Solvent was concentrated in vacuum to afford the crude title compound (0.35 g, crude) which was used as such for the next step.

Step f) Ethyl 1 -(tert-butoxycarbonylamino)-3-fluorocvclobutanecarboxylate (BB3-f)

To a solution of compound BB3-e (0.35 g, 0.0018 mol) in dry dioxane (6 mL) was added tert- butanol (1 .8 mL), diphenyl phosphoryl azide (0.56 g, 0.002 mol) and triethylamine (0.2 g, 0.002 mol) and the mixture was refluxed for 5 h. After completion of the reaction, EtOAc (60 mL) was added to the reaction mixture and the organic layer was washed with 5 % citric acid (2 x 20mL) followed by saturated NaHC0₃ (50 mL). The organic solvent was evaporated under reduced pressure. To the residue EtOAc (100 mL) was added and the the organic layer was washed with brine and dried over anhydrous Na₂S0₄. Solvent was concentrated in vacuum and the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 5-10 % EtOAc in pet ether) which afforded the title compound (0.27 g, 56 %).

Step q) tert-Butyl 3-fluoro-1 -(hvdroxymethyl)cvclobutylcarbamate (BB3-q)

To a ice cooled solution of compound BB3-f (0.27 g, 0.001 mol) in dry THF (10 mL) was slowly added a solution of 2M lithium borohydride (2 mL, 0.004 mol) and the mixture was allowed to warm up to room temperature. The mixture was stirred for 3 h at room temperature. The reaction mixture was quenched with ice water (2 mL) and 5 % citric acid (5 mL) and the crude product was extracted with DCM (2 x 50mL). The organic layer was washed with brine and dried over anhydrous Na₂S0₄. Solvent was concentrated in vacuum and the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 15-18 % EtOAc in p. ether) which afforded the title compound (90 mg, 39 %).

Step h) tert-Butyl 3-fluoro-1 -formylcvclobutylcarbamate (BB3-h)

To a degassed solution of compound BB3-g (90 mg, 0.0004 mol) in dry DCM (4.5 mL) was added Dess-Martin Periodinane (0.21 g, 0.0005 mol) and the mixture was stirred for 3 h at room temperature. EtOAc (30 mL) was added and the organic layer was washed with 10 % sodium thiosulphate solution (2 x 10 mL), 0.5 M NaOH (20 mL) and with brine. The organic layer was dried over anhydrous Na₂S0₄. Solvent was concentrated in vacuum and the crude product was purified by column chromatography (silica gel 60-120 mesh, eluent 10-15 % EtOAc in pet ether) which afforded the title compound (75 mg, 87 %).

Step i) tert-Butyl 1 -(2-(tert-butylamino)-1-hvdroxy-2-oxoethyl)-3-fluorocvclobutylcarbamate (BB3- D

To an ice cooled solution of compound BB3-h (1.3 g, 0.0059 mol) in dry DCM (25 mL) was added ie f-butyl isocyanide (0.75 g, 0.0089 mol) and dry pyridine (2.6 mL). Trifluoroacetic acid (0.9 mL, 0.01 18 mol) was added over a period of ten minutes maintaining the temperature at 0 °C. The reaction mixture was slowly warmed to room temperature and stirred for 16 h. EtOAc (50 mL) was added and the organic phase was washed twice with 5 % citric acid and brine. The organic phase was evaporated and the crude product was dissolved in THF (25 mL). 1 M LiOH solution in MeOH-H₂0 (3:2v/v) (2.6 mL) was added and the mixture was stirred for 2h at room temperature. The reaction mixture was quenched with 5 % citric acid and the mixture was extracted with ethyl acetate (2 x 25 mL). The organic layer was washed with brine and dried over anhydrous Na₂S0₄. Solvent was evaporated in vacuum and to afford the title compound which was pure enough to be used in the next step (1 .6 g, 84 %).

Step j) 2-(1 -(tert-Butoxycarbonylamino)-3-fluorocvclobutyl)-2-hvdroxyacetic acid (BB3)

Compound BB3-i (1.6 g, 5.0 mmol) was refluxed with 6N HCI (60 mL) for 16 h until the amide hydrolysis was complete. The solvent was evaporated under reduced pressure and co- evaporated several times with water. The product was dissolved in THF:H₂0 (7:3 v/v, 50 mL), cooled to 0 °C and Et₃N (2.1 mL, 15 mmol) was added followed by di-ie f-butyl dicarbonate (2.18 g, 10 mmol). The mixture was stirred at room temperature overnight (pH was monitored in a regular interval and kept ~1 1 throughout the reaction). The reaction mixture was neutralized with 1 N HCI and the product was extracted with EtOAc (2 x 50 mL). The organic layer was washed with brine and dried over anhydrous Na₂S0_4. The solvent was evaporated under reduced pressure followed by purification by column chromatography (silica gel 60-120 mesh, eluent 5 % MeOH in CHCI₃) which afforded the title P1 building block as a solid (0.65 g, 50%). ¹H N MR (400 MHz, d₆-DMSO) δ 7.01 (br s, 1 H), 5.16 (br m, 1 H), 4.97 (br m, 1 H), 2.49 (br m, 5H), 1 .36 (s, 9H); MS ES⁺ m/z 262 (100 %).

Building block 4 (BB4), a P1 building block

Step a R = TBDMS BB4-b BB4-C BB4

BB4-a, R = H Step a) tert-butyl 1 -(hvdroxymethyl)-3-methoxycvclobutylcarbamate (BB4-a)

500 mg (1 .51 mmol) of tert-butyl 1 -((tert-butyldimethylsilyloxy)methyl)-3- hydroxycyclobutylcarbamate (prepared by reduction of ethyl-1 [ [(tert-butyloxy)carbonyl] amino]- 3-hydroxycyclobutane-1 -carboxylate as described in J. Med. Chem., 1990 33(10) 2905-2915) and proton sponge (Ν,Ν,Ν',Ν' tetramethylnapthalene-1 ,8 diamine) (1 .63 g, 6.04 mmol) were dissolved in DCM (18 ml_), cooled down to 0 °C, and 447 mg (3.02 mmol) of trimethyloxonium borontetrafluoride was added in one portion as a solid under vigorous stirring. The reaction mixture was stirred for 3h and diluted with DCM (50 ml.) and brine (20 ml_), added under vigorous stirring. The organic phase was washed with sodium bicarbonate, brine, dried over sodium sulphate, evaporated and purified on short silica column (DCM as an eluent). The resulting product was dissolved in THF(5 ml_), and a solution of tetrabutylammonium fluoride in THF (1 M, 4.5 ml.) was added, and the reaction was stirred at room temperature for 4.5 h. The reaction was monitored by TLC and once deemed to have reached completion, it was absorbed onto silica and purified on silica (EtOAc-hexane 1 :1 to neat EtOAc) which gave the title compound (251 mg, 72%). LC/MS 232 (M+1 ).

Step b) tert-Butyl 1 -formyl-3-methoxycvclobutylcarbamate (BB4-b)

Alcohol BB4-a was dissolved in DCM (20 ml.) and Dess-Martin reagent was added in one portion. The reaction was stirred for 2.5 hours. Once the reaction was deemed to have reached completion, it was diluted with 50 ml. of DCM and 20 ml. of 10% Na₂S₂0₃ was added. The mixture was stirred, washed with sodium bicarbonate, brine, and the organic phase was dried over sodium sulphate. Purification on silica (EtOAc-hexane 1 :1 to neat EtOAc) gave the title compound (500 mg, 59%).

Step c) ri -(tert-Butylcarbamoyl-hvdroxymethyl)-3-methoxycvclobutyll-carbamic acid tert-butyl ester (BB4-c)

Aldehyde BB4-b, 4.45 mmol, was dissolved in dry DCM (1 1 ml_). Pyridine (2 ml.) was added under stirring conditions, followed by adding ie f-butyl isocyanide (6.68 mmol). The reaction was placed in an ice-bath and TFA (0.68 mL) was added dropwise during 20 min. The reaction mixture was stirred overnight. The reaction was then deemed to have reached completion and the solvent was removed under reduced pressure. The residue was redissolved in EtOAc and washed with 1 M HCI (2x), sodium bicarbonate, brine, and the organic phase dried over sodium sulphate and evaporated. The remaining residue was dissolved in dioxane and stirred with lithium hydroxide solution overnight and neutralized with citric acid. The product was extracted with EtOAc from the resulting solution and purified on silica (EtOAc-hexane 1 :3 to 1 :1 ) which gave the title compound (850 mg, 58%). Step d) (1 -tert-Butoxycarbonylarriino-3-methoxy-cvclobutyl)-hvdroxy-acetic acid (BB4)

The amide BB4-C (850 mg, 2.57 mmol) was refluxed with 6N HCI (60 mL) for 16 h until the amide hydrolysis was complete. The solvent was evaporated under reduced pressure and co- evaporated with water. The product was dissolved in THF:H₂0 (7:3 v/v, 50 mL), cooled to 0 °C and Et₃N (1.4 mL, 10.2 mmol) was added followed by di-ie f-butyl dicarbonate (2.25 g, 10.2 mol). The mixture was stirred at room temperature overnight. The reaction mixture was washed with EtOAc followed by acidifying to pH3 with 1 N HCI and extracted with EtOAc (2 x 50 mL). The organic layer was washed with brine and dried over anhydrous Na₂S0_4. The solvent was evaporated under reduced pressure which gave the title compound (360 mg, 51 %).

Building block 5, a P1 g-hydroxyamide

BB5

2-(1 -((Tert-butoxycarbonyl)amino)cvclopropyl)-2-hvdroxyacetic acid (BB5)

The title compound was prepared in 29% overall yield from N-Boc-1 - aminocyclopropanecarboxylic acid (5.03 g, 25.0 mmol), according to the procedure described for the preparation of BB1 .

Building block 6, a prime side amine (BB6)

1 -Methyl-1 H-imidazol-4-amine (BB6)

The title compound was achieved by catalytic hydrogenation in MeOH of 1 -methyl-4-nitro-1 H- imidazole using Pd/C as catalyst. Removal of the catalyst by filtration of the reaction mixture through Celite followed by evaporation of the solvent gave the title compound, which was used in the next step without further purification.

Building block 7, a P1 g-hvdroxyamide

BB7 The title compound was prepared from tert-butyl (l -formylcyclopentyl)carbamate according to the procedure described for the preparation of BB1.

Building block 8, Cyclopropylchloroformiate

Step a) Cyclopropyl benzoate (BB8-a)

Cyclopropyl benzoate was prepared in 98% yield from vinyl benzoate following Lorenz JC, et al. J Org Chem 2004, 69, 327.

Step b) Cyclopropyl alcohol (BB8-b)

The ester from the previous step (1 .09 g, 6.72 mmol) was suspended in phosphate buffer (0.1 M, pH 7.2, 40 mL), and Et₂0 (0.5 mL) was added. Candida lipase B (Novozym 435) immobilised on acrylic beads (0.5 g) was added and the mixture was shaken on a planar shaker for 48 h. 1 M aq NaOH (600 μί) solution was added after 12 h to adjust the pH from 6 to 7. The mixture was filtered through celite and diluted with H₂0 (50 mL). The solution was washed with hexane (40 mL), and the organic phase was discarded. The aqueous phase was saturated with NaCI and extracted with 5 X 20 mL Et₂0. The combined organic phases were washed with a 9:1 mixture of brine and aq sat NaHC0₃ (3 X 20 mL). The product was further purified by distillation at 200 mBar, 95 °C. This gave 245 mg of the title compound with some minor impurities, and the product was used as such in the next step.

Step c) Cyclopropyl chloroformiate (BB8)

The product of the previous step (50 mg, 0.86 mmol) was dissolved in DCM (1 mL) and cooled to 0 °C. Phosgene (20% wt in toluene, 452 μί, 0.86 mmol) was added followed by K₂C0₃ (357 mg, 2.58 mmol). The reaction was stirred vigorously for 2 h and the product was used as a solution in the next step (i.e. Step c, Method 2).

Oxetane-3-yl chloroformiate (BB9)

Oxetan-3-ol (39 mg, 0.53 mmol) was reacted with phosgene (1.9 M solution in toluene, 0.48 mmol, 255 μί) at 0 °C in DCM (1 .5 mL) in the presence of pyridine (0.53 mmol, 43 μί) for 5 minutes. The reaction solution was used directly in the next step (i.e. Step c, Method 2). Example 1 (Method 1 )

Step a) Tert-butyl (3-fluoro-1 -(1 -hydroxy-2-((1 -methyl-1 H-pyrazol-3-yl)amino)-2- oxoethvDcvclobutvDcarbamate (1 -a)

1 -Methyl-1 H-pyrazol-3-amine (1 eq.) and DIEA (4 eq) was added to a solution of the P1 - building block BB3 (1 eq) dissolved in DMF. The solution was cooled to 0 °C and after 10 minutes HATU (1 eq) was added. After approximately 2 hours at RT, LC-MS showed product and no starting material and the solvent was removed by rotary evaporation. The crude product was dissolved in 40 ml. EtOAc and washed with 25 ml. sat. NaHC0₃ (aq). The organic phase was dried with Na₂S0₄, filtered and evaporated to dryness. The crude product was purified by silica flash column chromatography, which gave the title compound.

Step b) Tert-butyl (1 -((3-fluoro-1 -(1 -hvdroxy-2-((1 -methyl-1 H-pyrazol-3-vnamino>2- oxoethyl)cvclobutyl)amino)-3-(1 -fluorocvclopentyl)-1 -oxopropan-2-yl)carbamate (1 -b)

The Boc-protected amine 1 -a (0.48 mmol), was treated with HCI (4M in dioxane, 5ml_) for 3h whereafter the reaction mixture was concentrated. The resulting hydrochloride salt of the amine was added to a cold solution of 2-((tert-butoxycarbonyl)amino)-3-(1 -fluorocyclopentyl)propanoic acid and HATU (200 mg, 0.53 mmol) in anh. DMF (4 ml.) was added at 0 °C, followed by addition of DIEA (335 μΙ_, 1.92 mmol). The reaction mixture was stirred at room temperature over night, and then concentrated. Purification by flash column chromatography (EtOAc//so- Hexane, 0:1 -7:3) gave the title compound (0.130 g, 70%). MS m/z 500.4 [M+H]⁺.

Step c) Tert-butyl (1 -((3-fluoro-1 -(2-((1 -methyl-1 H-pyrazol-3-yl)amino)-2- oxoacetyl)cvclobutyl)amino)-3-(1 -fluorocvclopentyl)-1 -oxopropan-2-yl)carbamate (1 )

Dess-Martin periodinane (0.301 g, 0.71 mmol) was added to a solution of the ohydroxy amide 1 -b (0.184 g, 0.47 mmol) in DCM (5 ml). The reaction mixture was stirred at room temperature over night and quenched with 10% (aq) Na₂S₂0₃ and (aq) NaHC0₃ (sat). The phases were separated and the organic layer was dried (Na₂S0₄) and concentrated. Purification by RP-LC- MS gave the title compound. MS m/z 498.4 [M+H]⁺.

Example 2 (Method 2)

Step a) Tert-butyl (1 -(1 -hvdroxy-2-((1 -methyl-1 H-pyrazol-3-yl)amino)-2- oxoethvDcvclopropyDcarbamate (2a)

1 -Methyl-1 H-pyrazol-3-amine (4 eq.) and DIEA (4.6 eq) was added to a solution of the P1 - building block BB5 (1 eq) dissolved in DMF. The solution was cooled to 0 °C and after 10 minutes HATU (1 eq) was added. After approximately 2 hours at RT, LC-MS showed product and no starting material and saturated NaHC0₃ was added and the solution was extracted with EtOAc. The organic phase was washed with NaCI(aq), dried with Na₂S0₄, filtered and evaporated to dryness. The afforded residue was purified by silica flash column chromatography, which gave the title compound (542 mg, 52%).

Step b) Tert-butyl (1 -((1 -(1 -hvdroxy-2-((1 -methyl-1 H-pyrazol-3-yl)amino)-2- oxoethyl)cvclopropyl)amino)-3-(1 -methylcvclopentyl)-1 -oxopropan-2-yl)carbamate (2b)

A solution of 4M HCI in 1 ,4-dioxane was added to a methanolic solution of the Boc protected amine 2a. The reaction mixture was stirred for 1 h and then concentrated. To the resulting amine (0.16 mmol) were added 2-tert-butoxycarbonylamino-3-(1 -methylcyclopentyl)-propionic acid (47.7 mg, 0.176 mmol, 1.1 eq), HATU (68.8 mg, 1 eq) and DIEA (0.12 ml) and DMF (1 .6 ml) at 0 °C whereafter the reaction mixture was allowed to attain room temperature. After 5h, saturated NaHC0₃ was added and the solution was extracted with EtOAc. The organic phase was washed with NaCI(aq), dried with Na₂S0₄, filtered and evaporated to dryness. The afforded residue was purified by silica flash column chromatography eluted with a gradient of

EtOAc/heptane, which gave the title compound in quantitative yield. MS m/z 464 [M+H]⁺.

Step c) Ethyl (1 -((1 -(1 -hydroxy-2-((1 -methyl-1 H-pyrazol-3-yl)amino)-2- oxoethyl)cvclopropyl)amino)-3-(1 -methylcvclopentyl)-1 -oxopropan-2-yl)carbamate (2c) Compound 2b (0.16 mmol) was dissolved in 4M HCI in dioxane (1.5 ml_). The solution was stirred for 20 min at room temperature and then concentrated under vacuum. The afforded residue was dissolved in DCM (3.5 ml) whereafter DIEA (50 μΙ_, 2 eq), ethyl chloroformate (23 μΙ_, 1.5 eq) and finally more DIEA(1.5 eq) were added. After 15 min, the reaction was quenched by addition of 5% citric acid (3.5 ml). The organic phase was washed with saturated NaHC0₃ (aq, (3,5 ml.) and then concentrated. MS m/z 436 [M+H].⁺ The crude compound was used in next step without further purification.

Step d) Ethyl (1-((1-(2-((1-methyl-1 H-pyrazol-3-yl)amino)-2-oxoacetyl)cvclopropyl)amino)-3-(1- methylcvclopentyl)-1 -oxopropan-2-yl Carbamate (2)

A solution of Dess Martin periodinane (105 mg, 0.25 mmol) in DCM (1 ml.) was added to the hydroxy amide 2c (0.16 mmol). After stirring for 1 h, the reaction was quenched by addition of a 1 :1 mixture of saturated NaHC0₃ (aq) and 10% Na₂S₂0₃. The layers were separated and the organic layer was washed with saturated NaCI and then concentrated. The afforded crude product was purified on a prep RP HPLC-UV/MS, which gave the title compound (14 mg). MS 434 [M+1] ⁺.

Examples 3-12

The compounds illustrated in the tables below were prepared according to the procedure described in Example 1 or 2, using the appropriate prime side, P1 and P2 building blocks, followed by oxidation to the end product a-keto amide.

¹ The [M-H]^" ion was observed.

O rfcr O N-A¹

Ex. Method R ΓΜ+ΗΓ n N-A¹

6 2 ethyl F 1 N-methylpyrazole 438

7 1 t.butyl F 1 N-methylimidazole 466.5

8 1 t.butyl F 1 N-methylpyrazole

9 1 t.butyl F 2 N-methylimidazole 480.1

10 2 ethyl F 2 N-methylimidazole 452.0

1 1 2 isopropyl F 2 N-methylimidazole 466.1

12 1 t.butyl F 4 N-methylpyrazole

The compound was isolated as a single diasteromere. Separation was achieved by preparative HPLC.

Biological Examples

Determination of cathepsin K proteolytic catalytic activity

Convenient assays for cathepsin K are carried out using human recombinant enzyme, such as that described in PDB.

ID BC016058 standard; mRNA; HUM; 1699 BP.

DE Homo sapiens cathepsin K (pycnodysostosis), mRNA (cDNA clone MGC:23107

RX MEDLINE;. RX PUBMED: 12477932.

DR RZPD: IRAI_p962G1234.

DR SWISS-PROT: P43235:

The recombinant cathepsin K can be expressed in a variety of commercially available expression systems including E coli, Pichia and Baculovirus systems. The purified enzyme is activated by removal of the prosequence by conventional methods.

Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically H-D-Ala-Leu-Lys-AMC, and were determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTA and 10 mM 2-mercaptoethanol or 100 mM Na phosphate, imM EDTA, 0.1 %PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20 mM cysteine, in each case optionally with 1 M DTT as stabiliser. The enzyme concentration used was 5 nM. The stock substrate solution was prepared at 10 mM in DMSO. Screens were carried out at a fixed substrate concentration of 60 μΜ and detailed kinetic studies with doubling dilutions of substrate from 250 μΜ. The total DMSO concentration in the assay was kept below 3%. All assays were conducted at ambient temperature. Product fluorescence (excitation at 390 nm, emission at 460 nm) was monitored with a Labsystems Fluoroskan Ascent fluorescent plate reader. Product progress curves were generated over 15 minutes following generation of AMC product.

Cathepsin S Ki determination

The assay uses baculovirus-expressed human cathepsin S and the boc-Val-Leu-Lys-AMC fluorescent substrate available from Bachem in a 384 well plate format, in which 7 test compounds can be tested in parallel with a positive control comprising a known cathepsin S inhibitor comparator.

Substrate dilutions

280 L/well of 12.5% DMSO are added to rows B - H of two columns of a 96 deep well polypropylene plate. 70[\Uwe\\ of substrate is added to row A. 2 x 250μΙ_Λ βΙΙ of assay buffer (100 mM Na phosphate, 100mM NaCI, pH 6.5) is added to row A, mixed, and double diluted down the plate to row H.

Inhibitor dilutions

100 μ-Jwell of assay buffer is added to columns 2-5 and 7-12 of 4 rows of a 96 well V bottom polypropylene plate. 200 L/well of assay buffer is added to columns 1 and 6.

The first test compound prepared in DMSO is added to column 1 of the top row, typically at a volume to provide between 10 and 30 times the initially determined rough K,. The rough K, is calculated from a preliminary run in which 10 μ-Jwell of 1 mM boc-VLK-AMC (1/10 dilution of 10 mM stock in DMSO diluted into assay buffer) is dispensed to rows B to H and 20 μΙ/well to row A of a 96 well Microfluor™ plate. 2 μΙ of each 10 mM test compound is added to a separate well on row A, columns 1 -10. Add 90 μΙ assay buffer containing 1 mM DTT and 2 nM cathepsin S to each well of rows B-H and 180 μΙ to row A. Mix row A using a multichannel pipette and double dilute to row G. Mix row H and read in the fluorescent spectrophotometer. The readings are Prism data fitted to the competitive inhibition equation, setting S = 100 μΜ and K_M = 100 μΜ to obtain an estimate of the K,, up to a maximum of 100 μΜ.

The second test compound is added to column 6 of the top row, the third to column 1 of the second row etc. Add 1 μΙ_ of comparator to column 6 of the bottom row. Mix column 1 and double dilute to column 5. Mix column 6 and double dilute to column 10.

Using an 8-channel multistepping pipette set to 5 x 10 μΙ_, distribute 10 μ-Jwell of substrate to the 384 well assay plate. Distribute the first column of the substrate dilution plate to all columns of the assay plate starting at row A. The tip spacing of the multichannel pipette will correctly skip alternate rows. Distribute the second column to all columns starting at row B.

Using a 12-channel multistepping pipette set to 4 x 10μΙ_, distribute 'l O UweW of inhibitor to the

384 well assay plate. Distribute the first row of the inhibitor dilution plate to alternate rows of the assay plate starting at A1 . The tip spacing of the multichannel pipette will correctly skip alternate columns. Similarly, distribute the second, third and fourth rows to alternate rows and columns starting at A2, B1 and B2 respectively.

Mix 20 ml. assay buffer and 20 μΙ_ 1 M DTT. Add sufficient cathepsin S to give 2 nM final concentration.

Using the a distributor such as a Multidrop 384, add 30 μ-Jwell to all wells of the assay plate and read in fluorescent spectrophotometer such as an Ascent.

Fluorescent readings, (excitation and emission wavelengths 390 nm and 460 nm respectively, set using bandpass filters) reflecting the extent of enzyme cleavage of the fluorescent substrate, notwithstanding the inhibitor, are linear rate fitted for each well.

Fitted rates for all wells for each inhibitor are fitted to the competitive inhibition equation using SigmaPlot 2000 to determine V, Km and Ki values.

Cathepsin L Ki

The procedure above with the following amendments is used for the determination of Ki for cathepsin L.

The enzyme is commercially available human cathepsin L (for example Calbiochem). The substrate is H-D-Val-Leu-Lys-AMC available from Bahcem. The assay buffer is 100mM sodium acetate 1 mM EDTA, pH5.5) The DMSO stock (10 mM in 100%DMSO) is diluted to 10% in assay buffer. Enzyme is prepared at 5 nM concentration in assay buffer plus 1 mM dithiothreitol just before use. 2μΙ_ of 10mM inhibitor made up in 100% DMSO is dispensed into row A. 10μΙ_ of 50 μΜ substrate (=1/200 dilution of 10 mM stock in DMSO, diluted in assay buffer).

Inhibition Studies

Potential inhibitors are screened using the above assay with variable concentrations of test compound. Reactions were initiated by addition of enzyme to buffered solutions of substrate and inhibitor. K, values were calculated according to equation 1.

where v₀ is the velocity of the reaction, V is the maximal velocity, S is the concentration of substrate with Michaelis constant of K_M, and / is the concentration of inhibitor.

The inhibition of cathepsin S, cathepsin K and cathepsin L exhibited by a selection of the compounds of the invention represented as Ki values in nM is presented in Table 1 . TABLE 1

Example Ki Cat. S Ki Cat. K Ki Cat. L

1 0.75 130 580

2 2.8 1800 2700

3 0.55 170 790

5 2.2 340 2100

8 3.3 360 2900

9 1.1 210 1 100

12 2.8 450 3200

The compounds of formula I are thus potent inhibitors of cathepsin S and yet selective over the closely related cathepsin K and L.

Permeability

This experiment measures transport of inhibitors through the cells of the human gastroenteric canal. The assay uses the well known Caco-2 cells with a passage number between 40 and 60.

Apical to basolateral transport

Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1 .5 mL and 0.4 mL transport buffer (TB), respectively, and the standard concentration of the tested substances is 10 μΜ. Furthermore all test solutions and buffers will contain 1 % DMSO. Prior to the experiment the transport plates are pre-coated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material. After 21 to 28 days in culture on filter supports, the cells are ready for permeability experiments.

Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denoted Wash, row 2 "30 minutes" and row 3 "60 minutes". Transport plate no 2 comprises 3 rows of 4 wells, one denoted row 4 "90 minutes", row 5 "120 minutes and the remaining row unassigned.

The culture medium from the apical wells is removed and the inserts are transferred to a wash row (No. 1 ) in a transport plate (plate no.1 ) out of 2 plates without inserts, which have already been prepared with 1.5 mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A→B screening the TB in basolateral well also contains 1 % Bovine Serum Albumin.

0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts and the cell monolayers equilibrated in the transport buffer system for 30 minutes at 37 °C in a polymix shaker. After being equilibrated to the buffer system the Transepithelial electrical resistance value (TEER) is measured in each well by an EVOM chop stick instrument. The TEER values are usually between 400 to 1000 Ω per well (depends on passage number used).

The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to the 30 minutes row (No. 2) and fresh 425 μΙ_ TB (pH 6.5), including the test substance is added to the apical (donor) well. The plates are incubated in a polymix shaker at 37 °C with a low shaking velocity of approximately 150 to 300 rpm.

After 30 minutes incubation in row 2, the inserts are moved to new pre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60 minutes), 4 (90 minutes) and 5 (120 minutes).

25 μΙ_ samples are taken from the apical solution after -2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.

300 μΙ_ will be taken from the basolateral (receiver) wells at each scheduled time point and the post value of TEER is measured at the end the experiment. To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at -20 °C until analysis by HPLC or LC-MS.

Basolateral to apical transport

Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1 .55 ml. and 0.4 ml. TB, respectively, and the standard concentration of the tested substances is 10 μΜ. Furthermore all test solutions and buffers will contain 1 % DMSO. Prior to the experiment the transport plates are precoated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material.

After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments. The culture medium from the apical wells are removed and the inserts are transferred to a wash row (No.1 ) in a new plate without inserts (Transport plate).

The transport plate comprises 3 rows of 4 wells. Row 1 is denoted "wash" and row 3 is the "experimental row". The transport plate has previously been prepared with 1 .5 ml. TB (pH 7.4) in wash row No. 1 and with 1 .55 ml. TB (pH 7.4), including the test substance, in experimental row No. 3 (donor side).

0.5 ml. transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts in row No. 1 and the cell monolayers are equilibrated in the transport buffer system for 30 minutes, 37 °C in a polymix shaker. After being equilibrated to the buffer system the TEER value is measured in each well by an EVOM chop stick instrument. The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to row 3 and 400 μΙ_ fresh TB, pH 6.5 is added to the inserts. After 30 minutes 250 μΙ_ is withdrawn from the apical (receiver) well and replaced by fresh transport buffer. Thereafter 250 μΙ_ samples will be withdrawn and replaced by fresh transport buffer every 30 minutes until the end of the experiment at 120 minutes, and finally a post value of TEER is measured at the end of the experiment. A 25 μΙ_ samples will be taken from the basolateral (donor) compartment after -2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.

To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at -20 °C until analysis by HPLC or LC-MS.

Calculation

Determination of the cumulative fraction absorbed, FA_cum, versus time. FA_cum is calculated from:

C RI

FA, cum c DI

Where C jj is the receiver concentration at the end of the interval i and Cpj is the donor concentration at the beginning of interval i. A linear relationship should be obtained.

The determination of permeability coefficients (P_app, cm/s) are calculated from:

_ (k - V_R )

P^APP ⁼ (A - 60) where k is the transport rate (min^"1) defined as the slope obtained by linear regression of cumulative fraction absorbed (FA_{CU M} ) as a function of time (min), VR is the volume in the receiver chamber (ml_), and A is the area of the filter (cm²). Reference compounds

Category of absorption in man Markers absorption in man (%)

PASSIVE TRANSPORT

Mannitol 16

Low (0-20%)

Methotrexate 20

Moderate (21-75%) Acyclovir 30

High (76-100%) Propranolol 90 Caffeine 100

ACTIVE TRANSPORT

Amino acid transporter L-Phenylalanine 100

ACTIVE EFFLUX

PGP-MDR1 Digoxin 30

Greater permeability through the gastrointestinal tissue is advantageous in that it allows for the use of a smaller dose to achieve similar levels of exposure to a less permeable compound administered in a higher dose. A low dose is advantageous in that it minimizes the cost of goods for a daily dose, which is a crucial parameter in a drug which is taken for protracted time periods.

All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims:

Claims

Claims Claims

1 . A compound of Formula I:

(I) wherein

one of A¹ and A² is N-CH₃ and the other is CH;

R¹ is CrC₆alkyl, CrC₆haloalkyl, C₃-C₆cycloalkyl or oxetan-3-yl, wherein C₃-C₆cycloalkyl is optionally substituted with one, two or three fluoro or with CF₃;

R^2a and R^2b are independently selected from H, halo, CrC₄alkyl, CrC₄haloalkyl, d-

C₄alkoxy;

R³ is CH₃ or F;

n is 1 , 2, 3 or 4

or a pharmaceutically acceptable salt, hydrate or N-oxide thereof.

2. A compound according to claim 1 , wherein R^2a and R^2b are both F.

3. A compound according to claim 1 , wherein one of R^2a and R^2b is H, and the other is F, CF₃ or MeO.

4. A compound according to any of claims 1 -5, wherein R^2a and R^2b are both H.

5. A compound according to claim 1 , wherein n is 1 or 2.

6. A compound according to any of claims 1 -7, wherein n is 2 and R^2a and R^2b are located in the 2-position of the cyclobutylenyl ring, i.e. having the structure Ic:

(Ic)

7. A compound according to claim 1 , wherein n is 1 and R and R are both H.

8. A compound according to any preceding claim, wherein R³ is CH₃.

9. A compound according to any preceding claim, wherein R³ is F.

10. A compound according to any preceding claim, wherein R¹ is CrC₄alkyl.

1 1 . A compound according to any preceding claim, wherein A¹ is N-CH₃ and A² is CH.

12. A pharmaceutical composition comprising a compound according to any preceding claim and a pharmaceutically acceptable vehicle therefor.

13. A compound according to any one of claims 1 -1 1 , for use in the treatment of a disorder characterised by inappropriate expression or activation of cathepsin S.

14. The compound according to claim 13, for use in the treatment of a disorder selected from a) Psoriasis;

b) Pruritus;

c) Autoimmune indications, including idiopathic thrombocytopenic purpura (ITP),

rheumatoid arthritis (RA), multiple schlerosis (MS), myasthenia gravis (MG), Sjogrens syndrome, Grave's disease and systemic lupus erythematosis (SLE); or

d) Non-automimmune indications including allergic rhinitis, asthma, artherosclerosis, chronic obstructive pulmonary disease (COPD) and chronic pain.

15. A method for the treatment of a disorder characterised by inappropriate expression or activation of cathepsin S, the method comprising the administration of an effective amount of a compound as defined in any of claims 1 -1 1 to a human or animal afflicted with or at risk of the disorder.

16. A method according to claim 15, wherein the disorder is

a) Psoriasis;

b) Pruritus;

c) An autoimmune indication selected from the group consisting of idiopathic

thrombocytopenic purpura (ITP), rheumatoid arthritis (RA), multiple schlerosis (MS), myasthenia gravis (MG), Sjogrens syndrome, Grave's disease and systemic lupus erythematosis (SLE); or A non-automimmune indication selected from the group consisting of allergic rhinitis, asthma, artherosclerosis, chronic obstructive pulmonary disease (COPD) and chronic pain.